Structural proteins of fish pancreatic disease virus and uses thereof

ABSTRACT

The present invention relates to the structural proteins of the causative agent of Pancreatic Disease in fish, nucleotide sequences encoding said proteins, vaccines comprising said proteins or nucleotide sequences and diagnostic kits comprising said proteins or nucleotide sequences.

FIELD OF THE INVENTION

The present invention relates to the structural proteins of thecausative agent of Pancreatic Disease in fish, nucleotide sequencesencoding said proteins, vaccines comprising said proteins or nucleotidesequences and diagnostic kits comprising said proteins or nucleotidesequences.

BACKGROUND OF THE INVENTION

Pancreatic Disease (PD) is a serious disease that affects fish, inparticular salmonid fish such as wild Atlantic salmon, rainbow trout andthe like. The disease causes lesions in the pancreas, including loss ofpancreatic exocrine tissue, and fibrosis, cardiac and skeletal musclemyopathies. Outbreaks of PD were first described in 1984 by Munro et al,in Helgoland Meeresuntersuchungen 37:571-586 (1984). PD typicallyaffects the fish post-molts during the first year after they aretransferred to sea sites and is reported to spread rapidly among farmfish held in sea cages. Clinical signs include lethargy with a tendencyto congregate in cage corners and to fail to maintain a horizontalposition, cessation of feeding (anorexia) and significant mortalities(Ferguson et al, J. Fish Disease 9:95-98, 1986). Murphy et al (in J.Fish Disease 15:401-408, 1992) confirmed these observations in a laterstudy, in which it was found that cardiac and skeletal myopathy isexacerbated in fish suffering from PD.

An outbreak of PD in a fish farm can cause growth to be reduced and upto 10 percent of surviving fish may prove to be runt. On Irish fishfarms PD causes significant mortality rates of 10 to 60 percent amongthe young fish during the first year after they are transferred to seasites (McLoughlin, M., Fish Farmer page 19, March/April 1995). Theestimated cost to the Irish industry in terms of loss of production iscurrently thought to be around £25 million per year. Consequently, thereis a great need for a vaccine for the prevention and/or treatment of PDin fish.

EP-A-712926 describes the isolation of the causative agent of PD fromtissues of PD affected fish and the identification of the virus as atoga-like virus. To prevent PD infections in fish, the use of attenuatedor inactivated PD for vaccination of the fish is accordingly suggested.A drawback in the production of inactivated vaccines from the PD virusdescribed in EP-A-712926 is the slow growth of the virus, in particularon cell cultures, which makes the manufacturing of said vaccines arelatively inefficient process. A further drawback with the inactivatedvaccines is the instability of the inactivated virus in the presence ofother inactivated pathogens resulting in potency loss. Fish vaccines aregenerally produced as multivalent vaccines, and significant higheramounts of inactivated virus are required in the multivalent vaccinethan would be necessary in a monovalent vaccine to compensate for theloss of potency.

SUMMARY OF THE INVENTION

The present invention provides the means to produce alternative vaccinesto prevent infection of fish with PD, in which the above mentioneddifficulties are overcome.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structural organisation of the various clonednucleotide sequences coding for the PD structural proteins.

FIG. 2 shows the nucleotide sequence of C-terminus of E2 gene/“long” 6Kgene/N-terminus of E1 gene. The putative cleavage sites between theE2/6K protein and 6K/E1 protein are represented by the vertical line(|)The nucleotide sequence encoding the “long” 6K protein is 204nucleotides long and encodes a protein of 68 amino acids. The numberingbetween brackets on the right of the sequence refers to the nucleotideand amino acid residues of the 6K gene or protein, respectively. Atnucleotide position 44 of the nucleotide sequence encoding the 6K gene,the G-residue can be replaced with an A residue, resulting in a 6Kprotein with an N residue at amino acid position 15 of the amino acidsequence depicted in the figure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the nucleotide sequence of the 3′part of the genomic RNA of a salmon PD virus (SPDV). This sequence of5179 nucleotides is depicted in SEQ ID NO 1 and contains several openreading frames (ORF's): On the coding strand nucleotide 2 to 1186 codesfor a non-structural protein, and another overlapping ORF starting fromnucleotide 997 to 5076 codes for the structural proteins. This ORF wasdesignated as p130. Other non-determined ORF's were found on the codingstrand (3447 to 3767 and 4289 to 4612) and the non coding strand (1207to 890, and 1232-837).

The ORF from nucleotide 2 to 1186 codes for the C-terminal part of anon-structural protein designated as NSP4; its deduced amino acid isdepicted in SEQ ID NO 2.

ORF p130 comprises the nucleotide sequences that encode the structuralproteins of the PD virus. The structural proteins of the PD virusconsist of a basic capsid protein, three envelope proteins designated asE1, E2 and E3, and a protein designated as the 6K protein. The aminoacid sequence of the whole protein encoded by the p130 ORF is depictedin SEQ ID NO 3. After processing, the p130 protein is spliced into thecapsid protein (aa 76-375 of p130), E3 (aa 358-428 of p130), E2(aa429-866 of p130), 6K (aa 867-898 of p130), and E1 (aa 899-1359 ofp130).

The nucleotide sequence encoding the capsid protein of the PD virus islocated at nucleotide 1222 to 2067 of SEQ ID NO 1. The correspondingamino acid sequence (total 282 amino acids) is depicted in SEQ ID NO 4.

The nucleotide sequence encoding the envelope proteins E3, E2 and E1 arelocated at nucleotides 2068-2280, 2281-3594 and 3691-5076 respectively,of the nucleotide sequence depicted in SEQ ID NO 1. The correspondingamino acid sequences of the E3, E2 and E1 proteins are depicted in SEQID No's 5, 6 and 8 respectively.

The nucleotide sequence encoding the 6K protein is located at nucleotide3595 to 3690 of the nucleotide sequence depicted in SEQ ID NO 1, and thecorresponding amino acid sequence of the 6K protein is depicted in SEQID NO 7. Further sequence analysis of the viral RNA extracted from PDinfected pancreas tissue revealed the existence of a longer variant ofthe 6K protein having 68 amino acids in length compared to the 6Kprotein of 32 amino acids depicted in SEQ ID NO 7. The nucleotidesequence (SEQ ID NO 14) encoding the longer variant of 6K protein is 204nucleotides in length compared to the 96 nucleotides of the nucleotidesequence encoding the truncated 6K protein. The nucleotide sequenceencoding the long variant of 6K protein and the deduced amino acidsequence thereof are shown in FIG. 2 and SEQ ID NO 14 and SEQ ID NO 15respectively.

The cloning and characterisation of the nucleotide sequences of thepresent invention provides for the production of the structural proteinsof the PD virus using standard recombinant DNA technology (Sambrooke etal., Molecular Cloning: a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1989). Cloning techniques andsubsequent protein expression using in vitro expression systems are wellknown in the art. In this way, recombinant structural PDV proteins canbe obtained, that are substantially free from other PDV proteins. Theseisolated structural proteins can be used to manufacture subunit vaccinesto protect against infection of PD in fish. The subunit vaccines may beused as marker vaccines in fish to distinguish vaccination from fieldinfections with PD. Alternatively the nucleotide sequences encoding thestructural proteins of the PD virus can be used to manufacture DNAvaccines or vector vaccines to protect against infection of fish withPD. The nucleotide sequences and recombinant PD proteins can furthermorebe used for diagnostic purposes, for instances to detect the presence ofPD virus in the field or anti-PD antibodies in fish. Additionally, therecombinant PD proteins of the present invention can be used to producePD specific antibodies. These antibodies can also be used for diagnosticpurposes such as the detection of PD virus in fish or in the field.

Thus, in a first aspect the invention provides for a nucleic acidcomprising the nucleotide sequence depicted in SEQ ID NO 1 encoding thestructural proteins and part of NSP4 of the PD virus, fragments of saidnucleotide sequence and a nucleic acid comprising the nucleotidesequence depicted in SEQ ID NO 14. Preferred fragments of the nucleotidesequences according to the invention are nucleotide fragments 1222-5076(also referred to as p130 encoding the capsid, E3, E2, 6K and E1proteins), 2068-5076 (also referred to as p98 encoding the E3, E2, 6Kand E1 proteins), 2068-3594 (also referred to as pE2 encoding E3 and E2proteins), 1222-2067 (capsid), 2068-2280 (E3), 2281-3594 (E2), 3595-3690(6K), and 3691-5076 (E1). For the purpose of this invention thenucleotide sequences according to the present invention also encompassthe nucleotide sequence depicted in SEQ ID NO 1 and fragment sequencesthereof (such as the p130 and p98 fragments) which at least comprise anucleotide sequence encoding for a 6K protein, wherein the nucleotidesequence depicted by nucleotide 3595-3690 of SEQ ID NO 1 has beensubstituted with the nucleotide sequence depicted in SEQ ID NO 14.

Also within the scope of this invention are nucleotide sequencescomprising tandem arrays of the nucleic acid comprising the sequencedepicted in SEQ ID NO 1 or SEQ ID NO 14 or fragments thereof. Nucleotidesequences that are complementary to the sequence depicted in SEQ ID NO1, SEQ ID NO 14, or parts thereof are also within the scope of theinvention, as well as nucleotide sequences that hybridise with thesequence depicted in SEQ ID NO 1 or SEQ ID NO 14. The hybridisationconditions for this purpose are stringent, preferably highly stringent.According to the present invention the term “stringent” means washingconditions of 1×SSC, 0.1% SDS at a temperature of 65° C.; highlystringent conditions refer to a reduction in SSC towards 0.3×SSC.

Nucleotide sequences that hybridise with the sequence shown in SEQ ID NO1 or SEQ ID NO 14 are understood to be nucleotide sequences that have asequence homology of at least 70%, preferably 80%, more preferably 90%with the corresponding matching part of the sequence depicted in SEQ IDNO 1 or SEQ ID NO 14. According to the present invention the sequencehomology is determined by comparing the nucleotide sequence with thecorresponding part of the sequence depicted in SEQ ID NO 1 or SEQ ID NO14. The sequence homology between a nucleotide and the sequence in SEQID NO 1 or SEQ ID NO 14 can be determined via common sequence analysisprogram such as BLASTN and the like. The optimal match area isautomatically determined by these programs. Homologous sequences caneasily be isolated from closely related PD virus strains with thesequence depicted in SEQ ID NO 1 or SEQ ID NO 14 or fragments of thesesequences using routine cloning and hybridisation techniques. SleepingDisease (SD) virus is closely related to PD virus and the nucleic acidsequences encoding the structural capsid, E3, E2, E1 and 6K proteins ofSD virus have the necssary sequence homology with the nucleic acidsequences depicted in SEQ ID NO 1 and 14. Thus these SD nucleic acidsequences are also within the present invention.

The nucleotide sequences of the invention can be used in the preparationof a DNA vaccine to vaccinate fish against PD infection. DNA vaccinationrefers to the induction of an immune response to one or more antigensthat are expressed in vivo from a gene inserted in a DNA plasmid whichhas been inoculated directly into the vaccinated fish. Thus in a secondaspect of the invention there is provided for a DNA vaccine comprising apharmaceutically acceptable carrier and a DNA plasmid in which anucleotide sequence encoding one or more PDV structural proteins isoperably linked to a transcriptional regulatory sequence.

Preferably the nucleotide sequence to be used in the DNA plasmid is anucleotide sequence comprising the nucleotide sequence depicted in SEQID NO 1 or a nucleotide sequence comprising the nucleotide sequencedepicted in SEQ ID NO 14 or fragments of said nucleotide sequences.Preferred fragments of the nucleotide sequence depicted in SEQ ID NO 1or 14 are nucleotide fragments 1222-5076, 2068-5076, 2068-3594,1222-2067, 2068-2280, 2281-3594, 3595-3690 3691-5076 of the sequencedepicted in SEQ ID NO 1, and combinations thereof such as for example,fragment 1222-2067 with fragment 2281-3594. Also suitable for use in theDNA plasmid are nucleotide sequences that are complementary to thesequence of SEQ ID NO 1 or SEQ ID NO 14 or nucleotide sequences of whichthe sequence homology with the sequence depicted in SEQ ID NO 1 or SEQID NO 14 is at least 70%, preferably 80%, and more preferably 90%. Thesequence homology between the nucleotide sequences that are suitable foruse in the DNA plasmid is determined as described earlier.

DNA plasmids that are suitable for use in a DNA vaccine according to theinvention are conventional cloning or expression plasmids for bacterial,eukaryotic and yeast host cells, many of which are commerciallyavailable. Well known examples of such plasmids are pBR322 and pcDNA3(Invitrogen). The DNA plasmids according to the invention should be ableto induce protein expression of the nucleotide sequences. The DNAplasmid can comprise one or more nucleotide sequences according to theinvention. In addition, the DNA plasmid can comprise other nucleotidesequences such as the immune-stimulating oligonucleotides havingunmethylated CpG dinucleotides, or nucleotide sequences that code forother antigenic proteins or adjuvating cytokines.

Transcriptional regulatory sequences that are suitable for use in a DNAplasmid according to the invention comprise promoters such as the(human) cytomegalovirus immediate early promoter (Seed, B. et al.,Nature 329, 840-842, 1987; Fynan, E. F. et al., PNAS 90,11478-11482,1993; Ulmer, J. B. et al., Science 259, 1745-1748, 1993),Rous sarcoma virus LTR (RSV, Gorman, C. M. et al., PNAS 79, 6777-6781,1982; Fynan et al., supra; Ulmer et al., supra), the MPSV LTR (Stacey etal., J. Virology 50, 725-732, 1984), SV40 immediate early promoter(Sprague J. et al., J. Virology 45, 773,1983), the metallothioneinpromoter (Brinster, R. L. et al., Nature 296, 39-42, 1982), the majorlate promoter of Ad2, the β-actin promoter (Tang et al., Nature 356,152-154, 1992). The regulatory sequences may also include terminator andpolyadenylation sequences. Amongst the sequences that can be used arethe well known bovine growth hormone polyadenylation sequence, the SV40polyadenylation sequence, the human cytomegalovirus (hCMV) terminatorand polyadenylation sequences.

The DNA plasmid comprising a nucleotide sequence according to thepresent invention operably linked to a transcriptional regulatorysequence for use in the vaccine according to the invention can be nakedor can be packaged in a delivery system. Suitable delivery systems arelipid vesicles, Iscoms, dendromers, niosomes, polysaccharide matrices,and the like. Also very suitable as delivery system are attenuated livebacteria such as Salmonella.

The nucleotide sequences according to the invention can additionally beused in the production of a vector vaccine to vaccinate fish against PD.A vector vaccine is understood to be a vaccine in which a live,attenuated bacteria or virus has been modified so that it contains oneor more heterologous nucleotide sequences inserted into its geneticmaterial. These so called vector bacteria or viruses are capable ofcoexpressing the heterologous proteins encoded by the insertednucleotides. Thus in a third aspect the invention provides for a vectorvaccine comprising a live attenuated bacteria or virus which have beenmodified to comprise in their genetic material one or more of thenucleotide sequences of the present invention. Very suitable for use asa vaccine vector are, for example, vaccinia virus or Semliki forestvirus

The nucleotide sequences according to the invention can also be used forthe recombinant production of structural PD proteins, substantially freefrom other PD proteins. Thus in a fourth aspect the invention providesfor the structural proteins from PD virus. More specifically theinvention provides for a PD capsid protein, the PD envelope proteins E1,E2, and E3, and the 6K protein. In particular, there is provided for acapsid protein having the amino acid sequence depicted in SEQ ID NO 4 ora derivative thereof, an E3 protein having the amino acid sequencedepicted in SEQ ID NO 5 or a derivative thereof, an E2 protein havingthe amino acid sequence depicted in SEQ ID NO 6 or a derivative thereof,an E1 protein having the amino acid sequence depicted in SEQ ID NO 8 ora derivative thereof, and a 6K protein having the amino acid sequencedepicted in SEQ ID NO 7, SEQ ID NO 15 or a derivative thereof.

Derivative proteins are understood to be proteins which have alterationsin the amino acid sequencers) of the present invention which do notaffect the antigenic and/or immunogenic characteristics of theseproteins, that is, these derivative proteins are still capable ofinducing the production of antibodies that recognise and (cross)reactwith the PD virus and/or inducing an immune response in fish thatprotects against PD infection. Antigenic characteristics are understoodto be the ability to induce production of antibodies that recognise and(cross)-react with the PD virus. Immunogenic characteristics areunderstood to be the ability to induce an immune response in fish thatprotects against infection with PD. The alterations that can occur in asequence according to the present invention could, for instance, resultfrom conservative amino acid substitutions, deletions, insertions,inversions or additions of (an) amino acid(s) in the overall sequence.Amino acid substitutions that are expected not to alter theimmunological properties have been described. Amino acid replacementsbetween related amino acids or replacements which have occurredfrequently in evolution are, inter alia Ser/Ala, Ser/Gly, Asp/Gly,Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence andstructure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Based on this information, Lipman and Pearson developed amethod for rapid and sensitive protein comparison (Science, 1985, vol.227, 1435-1441) and determining the functional similarity betweenproteins and peptides having sequence homology. The derivative proteinsaccording to the invention are still capable to induce the production ofantibodies that recognise and (cross)-react with the PD virus and/or toinduce an immune response in the fish that protects against PDinfection. The capsid, E1, E2, E3, and 6K proteins derived from SleepingDisease (SD) virus are such derivative proteins according to theinvention. These proteins have an amino acid sequence that is identicalor almost identical to those of the PD virus as depicted in SEQ ID NO 4to 8 or 15. These proteins are capable to raise antibodies thatrecognize and cross-react with PD virus as well as SD virus. Otherderivatives are protein fragments that are still capable to induce theproduction of antibodies that recognise and (cross)-react with the PDvirus and/or to induce an immune response in the fish.

The proteins according to the invention can be prepared via standardrecombinant protein expression techniques. For this purpose a nucleotidesequence encoding one or more of the proteins according to the inventionor a multimere of said protein is inserted into an expression vector.Preferably the nucleotide sequence is a nucleotide sequence comprisingthe nucleotide sequence depicted in SEQ ID NO 1 or SEQ ID NO 14 or oneor more fragments of these sequences. Preferred fragments of thenucleotide sequences according to the invention are nucleotide fragments1222-5076, 2068-5076, 2068-3594, 1222-2067, 2068-2280, 2281-3594,3595-3690 3691-5076 of the sequence depicted in SEQ ID NO 1, andcombinations thereof such, for example, fragment 1222-2067 with fragment2281-3594. Further preferred fragments according to the invention arefragments of the nucleotide sequence depicted in SEQ ID NO 15 such asfor example the nucleotide sequence depicted by nucleotides 3595-3690 ofSEQ ID NO 1. Also suitable are nucleotide sequences that arecomplementary to the sequence of SEQ ID NO 1 or SEQ ID NO 14 ornucleotide sequences of which the sequence homology with the sequencedepicted in SEQ ID NO 1 or SEQ ID NO 14 is at least 70%, preferably 80%,and more preferably 90%. The sequence homology between the nucleotidesequences that are suitable for use in the DNA plasmid is determined asdescribed earlier.

Suitable expression vectors are, amongst others, plasmids, cosmids,viruses and YAC's (Yeast Artificial Chromosomes) which comprise thenecessary control regions for replication and expression. The expressionvector can be brought to expression in a host cell. Suitable host cellsare, for instance, bacteria, yeast cells and mammalian cells. Suchexpression techniques are well known in the art (Sambrooke et al.,Molecular Cloning: a Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1989). The expressed proteins can be isolatedand purified from the medium. Expression of the whole p130 ORF(nucleotide fragment 997 to 5076 of SEQ ID NO 1) might lead to theforming of virus-like particles due to the spontaneous assemblance ofthe structural proteins.

The invention furthermore provides for a vaccine comprising one or moreof the structural PD proteins and a pharmaceutically acceptable carrier.More specifically, a vaccine according to the invention comprises acapsid protein having an amino acid sequence depicted in SEQ ID NO 4 ora derivative thereof, an E3 protein having an amino acid sequencedepicted in SEQ ID NO 5 or a derivative thereof, an E2 protein having anamino acid sequence depicted in SEQ ID NO 6 or a derivative thereof, anE1 protein having an amino acid sequence depicted in SEQ ID NO 8 or aderivative thereof, a 6K protein having an amino acid sequence depictedin SEQ ID NO 7 or SEQ ID NO 15 or a derivative thereof, or a mixturecomprising two or more of the proteins according to the invention.Preferably the vaccine according to the invention comprises the E2protein, and optionally the capsid protein. Also preferred is a vaccinecomprising all structural proteins of PD; these proteins canspontaneously form virus-like particles, thus providing a vaccine thatclosely resembles that of the whole pathogen. Vaccines according to theinvention are suitable for use as a marker vaccine to distinguishbetween vaccination and infection by PD in the field. A preferredvaccine according to the invention is a marker vaccine comprising a 6Kprotein having the amino acid sequence depicted in SEQ ID NO 7.

A vaccine according to the invention can be prepared according totechniques well known to the skilled practitioner. General techniquesfor the preparation of DNA vaccines have been widely described, forexample in EP patent 0 773 295 and U.S. Pat. No. 5,580,859.

Vaccines according to the invention comprise an effective amount of theafore-mentioned DNA plasmids, vector bacteria or virus, or proteins anda pharmaceutically acceptable carrier. The term “effective” as usedherein is defined as the amount sufficient to induce an immune responsein the target fish. The amount of plasmid, vector or protein will dependon the type of plasmid or vector, the route of administration, the timeof administration, the species of the fish as well as age, generalhealth and diet.

In general, a dosage of 0.01 to 1000 μg protein per kg body weight,preferably 0.5 to 500, and more preferably 0.1 to 100 μg protein can beused. With respect to the DNA vaccines, generally a minimum dosage of 10pg. up to dosages of 1000 μg of plasmid have been described to besufficient for a suitable expression of the antigens in vivo.

Pharmaceutically acceptable carriers that are suitable for use in avaccine according to the invention are sterile water, saline, aqueousbuffers such as PBS and the like. In addition, a vaccine according tothe invention may comprise other additives such as adjuvants,stabilisers, anti-oxidants and others.

Suitable adjuvants include, amongst others, aluminium hydroxide,aluminium phosphate, amphigen, tocophenols, monophosphenyl lipid A,muramyl dipeptide, oil emulsions, glucans, carbomers, block-copolymers,cytokines and saponins such as Quil A. The amount of adjuvant addeddepends on the nature of the adjuvant itself.

Suitable stabilisers for use in a vaccine according to the inventionare, for example, carbohydrates including sorbitol, mannitol, starch,sucrose, dextrin, and glucose, proteins such as albumin or casein, andbuffers like alkaline phosphates.

The vaccines according to the invention are administered to the fish viainjection, spray, immersion or peroral. The administration protocol canbe optimised in accordance with standard vaccination practice.

The nucleotide sequences and the proteins according to the invention arealso suitable for use in diagnostics. The nucleotide sequences orfragments thereof can be used to detect the presence of PD virus in thefish. A primer spanning the C-terninal part of E2/6K/N-terminal part ofE1 (see FIG. 2) was used in RT-PCR to succesfully detect the presence ofPD virus in a clinical specimen of a PD outbreak. The proteins can beused to detect the presence of antibodies in the fish.

The proteins according to the invention can additionally be used for theproduction of antibodies, using the general techniques available to thepractitioner in the field. Preferably the proteins are used to producespecific monoclonal antibodies. The obtained antibodies may be utilisedin diagnostics, to detect PD virus in the field, or in the fish.

Thus, in another aspect, the present invention provides for a diagnostickit comprising one or more nucleotide sequences according to theinvention, or one or more structural proteins according to theinvention, or antibodies obtained with said proteins. Antibodiesaccording to the invention can be prepared according to standardtechniques. Procedures for immunising animals, e.g. mice with proteinsand selection of hybridomas producing immunogen specific monoclonalantibodies are well known in the art (see for example Coligan et al.(eds), Current protocols in Immunology, 1992; Kohler and Milstein,Nature 256:495-497, 1975; Steenbakkers et al., Mol. Biol. Rep.19:125-134, 1994).

The following examples are to illustrate the invention and should not beinterpreted to limit the invention in any way.

FIG. 1: structural organisation of the various cloned nucleotidesequences coding for the PD structural proteins.

FIG. 2: Nucleotide sequence of C-terminus of E2 gene/“long” 6Kgene/N-terminus of E1 gene. The putative cleavage sites between theE2/6K protein and 6K/E1 protein are presented by the vertical line(|)The nucleotide sequence encoding the “long” 6K protein is 204nucleotides long and encodes a protein of 68 amino acids. The numberingbetween brackets on the right of the sequence refers to thenucleotide-and amino acid residues of the 6K gene or proteinrespectively. At nucleotide position 44 of the nucleotide sequenceencoding the 6K gene the G-residue can be replaced with an A residue,resulting in a 6K protein with an N residue at amino acid position 15 ofthe amino acid sequence depicted in the figure.

EXAMPLES

Cells and Virus

Isolation and cultivation of a salmon PD virus (SPDV) strain was carriedout in general as described in EP-A-712926. The F93125 isolate of SPDVwas grown in Chinook salmon embryo (CHSE-214) cells as previouslydescribed (R. T. Nelson et al. (1995) Isolation of toga-like virus fromfarmed Atlantic salmon Salmo salar with pancreas disease. Diseases ofAquatic Organisms 22, pp. 25-32). For virus purification purposes,monolayer cultures of CHSE-214 grown to ˜80% confluence in 75 cm² flaskswere infected with 1 ml virus to give a multiplicity of infection of ˜1.After 1 hr adsorption an additional 14 ml supplemented Eagle's minimalessential medium (MEM) was introduced to each flask. The virus infectedflasks were incubated at 15° C. for 7 or 8 days, when virus-inducedcytopathic effect was evident, and the supernatant was collected.

Virus Purification

The supernatant (typically 500 ml from virus-infected cells wasclarified at 3000 g for 20 min. Polyethyleneglycol (PEG) and NaCl wereadded to give final concentrations of 6% and 2.2% respectively.Following overnight incubation at 4° C. the PEG precipitate wascollected by centrifugation for 2 h at 3000 g. The resultant pellet wasresuspended in PBS (1-2 ml) and, after clarification at 1000 g for 5minutes, the crude virus suspension was fractionated by equilibriumdensity centrifugation using 11 ml gradients (20-60% w/w in PBS) ofsucrose. After centrifugation for 18 hr at 75000 g at 4° C., 1 mlfractions were collected from the bottom of the gradient. Fractionscontaining virus were identified by immunoblotting using an PD-specificmouse monoclonal antibody (Welsh et al., submitted 1999).

Production of PD Virus cDNA Clones

Viral RNA was extracted from gradient-purified PD virus andvirus-infected cells using RNA isolator (Genosys) and stored as ethanolprecipitates. A cDNA library was made by random priming with RNAextracted from gradient-purified virus. This library consisted of clonescontaining inserts (250-500 bp) in the vector pUC18 (Sureclone ligationKit, Pharmacia). Clones were selected randomly from the library andfollowing sequencing and analysis using the BLAST program (University ofWisconsin, Genetics Computer Group) were mapped to the alphavirusgenome. The sequences of three clones, N11, N38 and N50, were used todesign oligonucleotide primers that were used in reversetranscription-polymerase chain reaction (RT-PCR) to amplify 3overlapping fragments encompassing the 5.2 kb region at the 3′terminusof the PD genome. The incorporation of Not I sites into the primersfacilitated the restriction ligation of two of these fragments into theNot I site of vector pBluescript (Stratagene). PCR was carried out usingExpand Long Template PCR System (Boehringer Mannheim) at 94° C. for 30s60° C. for 30s, 68° C. for 2 min. Another clone was produced using3′RACE (M. A. Frohmann et al., 1998; Rapid production of fill-lengthcDNA's from rare transcripts using a single gene-specificoligonucleotide primer. Proc. Natl. Acad. Sci.USA. 85, pp. 8998-9002).The reaction was performed using a 5′/3′ RACE kit (Boehringer Mannheim)with some modifications. Thus, RNA from gradient-purified virus wasindependently subjected to first-strand synthesis and the resultantcDNA's were amplified by PCR at 94° C. for 30s, 60° C. for 30s, 68° C.for I min.

Sequencing of PD Virus cDNA Clones

Cycle sequencing was performed using the ABI PRISM dye terminator readyreaction kit on purified plasmid DNA following the manufacturersprotocol (Perkin Elmer Cetus). Electropherograms were interpreted usingthe Sequence Navigator software (Perkin Elmer Cetus). The completenucleotide sequence of the 3′terminal 5.2 kb region of the PD virus RNAis presented in SEQ ID NO1.

An RT-PCR and sequence analysis using primers flanking the C-termninusof E2 and the N-terminus of E1 for viral RNA extracted directly from PDinfected pancreas tissue revealed a longer 6K-encoding nucleotidesequence than the one depicted by nucleotides 3595-3690 of SEQ ID NO 1.The nucleic acid encoding the full-length 6K protein as well as thededuced amino acid sequence are shown in FIG. 2.

SPDV pFastBac1 and pcDNA3.1(+) Constructs

Using standard cloning techniques (Sambrooke et al., Molecular Cloning:a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1989) four clones representing the SPDV structural region havebeen created in the vector pFastBac1 (Gibco BRL) for expression in thebaculovirus system. These clones have also been created in theexpression vector pcDNA3.1 (Invitrogen) for monoclonal antibodycharacterisation and use as a DNA vaccine. Details of how these cloneshave been produced are as follows:

Clone 1.

p130 encodes the complete structural gene region from the 1st ATG of thecapsid protein to the poly(A) tract (3944nt). cDNA was produced fromviral RNA by RT-PCR using the following primers:

5′ forward primer (5′130Not1): 5′-TGC ATG CGG CCG CAT GTT TCC CAT GCAATT CAC CAA C-3′ (SEQ ID NO 9)

3′ inverse primer (3′130Not1) (sequence 5′ to 3′): 5′-TGC ATG CGG CCGCTT GTA TTG AAA ATT TTA AAA CCA A-3′ (SEQ ID NO 10)

These primers contain a 5 nucleotide stretch (ensures restriction enzymerecognition) followed by a Not1 site then the appropriate SPDV sequence(highlighted in the attached sequence, from 1222 to 1245 for 5′130Not1and from 5143 to 5166 for 3′130Not1). The 3944nt cDNA product was clonedinto the Not1 site in both pFastBac1 and pcDNA3.1.

Clone 2.

p98 encodes for E3, E2, 6K and E1 to the poly(A) tract (3098nt). cDNAwas produced from viral RNA by RT-PCR using the following primers:

5′ forward primer (5′E3Not1): 5′-TGC ATG CGG CCG CAT GAC ACG CGC TCC.GGC CCT CCT G-3′ (SEQ ID NO 11)

3′ inverse primer (3′130Not1): 5′-TGC ATG CGG CCG CTT GTA TTG AAA ATTTTA AAA CCA A-3′ (SEQ ID NO 10)

The primer 5′E3Not1 contains a 5 nucleotide stretch (ensures restrictionenzyme recognition) followed by a Not1 site, an ATG (artificial startcodon) then the appropriate SPDV sequence (from 2067 to 2088) The primer3′130Not1 is as described above in Clonel. The 3098nt cDNA product wascloned into the Not1 site in both pFastBac1 and pcDNA3.1.

Clone 3.

pE2 encoding the E3 and E2 glycoproteins (1527nt). cDNA was producedfrom viral RNA by RT-PCR using the following primers:

5′ forward primer (5′E3Not1): 5′-TGC ATG CGG CCG CAT GAC ACG CGC TCC GGCCCT CCT G-3′(SEQ ID NO 11)

3′ inverse primer (3′E2Not1): 5′-TGC ATG CGG CCG CTC ACG CGC GAG CCC CTGGTA TGC AAC A-3′ (SEQ ID NO 12)

The primer 5′E3Not1 is as described above in Clone2. The primer 3′E2Not1contains a 5 nucleotide stretch (ensures restriction enzyme recognition)followed by a Not1 site, a TGA (artificial stop codon) then theappropriate SPDV sequence (highlighted in the attached sequence, from3571 to 3594). The 1527nt cDNA product was cloned into the Not1 site inboth pFastBac1 and pcDNA3.1.

Clone 4.

E2 encoding the E2 glycoprotein (1314nt). cDNA was produced from viralRNA by RT-PCR using the following primers:

5′ forward primer (5′E2Not1): 5′-TGC ATG CGG CCG CAT GGC TGT GTC TACGTCGCCTGC C-3′ (SEQ ID NO 13)

3′ inverse primer (3′E2Not1): 5′-TGC ATG CGG CCG CTC ACG CGC GAG CCC CTGGTA TGC AAC A-3′ (SEQ ID NO 12).

The primer 5′E2Not1 contains a 5 nucleotide stretch (ensures restrictionenzyme recognition) followed by a Not1 site, an ATG (artificial startcodon) then the appropriate SPDV sequence (from 2281 to 2301). Theprimer 3′E2Not1 is as described above in Clone 3. The 1314nt cDNAproduct was cloned into the Not1 site in both pFastBac1 and pcDNA3.1.

Insect cells (SF-9)were infected with the four recombinant baculovirusconstructs. Using monoclonals that were raised against whole-inactivatedPD virus, an IFT staining was performed on these recombinant baculovirusinfected SF-9 cells. All produced proteins reacted positively with themonoclonals, indicating that the recombinant proteins possess thewild-type epitopes.

Challenge Experiments

The proteins produced by all four constructs were collected using Tritonextraction. The proteins were BPL inactivated to prevent possible spreadof surviving recombinant baculoviruses in the environment. The proteinswere formulated into water-in-oil based vaccine formulations andinjected in a 0.2 ml vaccine volume.

ELISA analysis using anti-PD-E2 monoclonals (2D9 capture and 7A2) showedthat the amount of reactive epitopes per dose recombinant vaccine wascomparable or even higher than the amount of epitopes found in a dose ofthe conventional inactivated PD virus vaccine.

A standardised challenge experiment performed at 8 weekspost-vaccination in Atlantic salmon fish showed that protection againstchallenge with salmon PD virus could be obtained with these recombinantsub-unit vaccines. In the experiment, lesions in pancreas, skeletalmuscle and heart muscle were scored in the ordinary way. Significantlevels were calculated from Kruskal-Wallis one-way analysis of variance(non-parametric test). The vaccine formulation comprising the E2 orE2-E3 proteins gave similar levels of protection as obtained by theinactivated PD virus vaccine, while vaccines containing the recombinantproteins resulting from the p130 and p98 constructs, respectively, wereless protective then the PD virus vaccine.

Production of Antibodies.

DNA vaccination with proteins obtained from expression of the p130nucleotide construct was carried out in mice to test for the antigenicproperties of the recombinant proteins. After two intramuscularinoculations with p130-pcDNA3.1 recombinant expression plasmids (seeclone 1), the sera of mice showed an antibody reaction with in vitroproduced PD virus.

What is claimed is:
 1. An isolated structural protein of Fish PancreaticDisease virus, wherein the protein is a capsid protein, wherein theprotein comprises an amino acid sequence comprising SEQ ID NO:4.
 2. Apharmaceutical composition, comprising: the isolated protein of claim 1and a pharmaceutically acceptable carrier.
 3. A vaccine, comprising: theisolated structural protein of claim 1 and a pharmaceutically acceptablecarrier.
 4. A diagnostic kit, comprising: the isolated protein of claim1.